VisiGen Biotechnologies, Inc. is developing a sequencing system in which polymerase and nucleotides act together as direct molecular sensors of DNA base identity. More specifically, the technology detects the interaction between a modified nucleotide and a fluorescently-modified polymerase. As a nucleotide is incorporated into the growing DNA polymer, energy transfers from an excited donor fluorophore within the polymerase to an acceptor fluorophore within the nucleotide, stimulating the emission of a base-specific incorporation signature that is directly detected in real-time. Cutting-edge technologies, including single molecule detection, fluorescent molecule chemistry, computational biochemistry, and biomolecule engineering, are combined to create this novel sequencing system. DNA samples will be processed in massively parallel arrays that will enable large genomes to be sequenced in less than a day for approximately $1000 and permit sufficient oversampling to produce redundant data that will minimize errors. The system is being developed to identify pathogens (or variations thereof) and to enable comprehensive genome analysis. Proof of principle for VisiGen's technology is demonstrated: sequence information has been detected at the single molecule level in real-time using an immobilized, donor-labeled polymerase and 2 different acceptor-labeled dNTPs. Given the success of our federally-funded efforts, further development of the VisiGen system to sequence arrays of single DNA molecules in real-time is clearly warranted. The specific aims of the proposed research are to refine the sequencing chemistry and to design, build and test the next generation single-molecule DNA sequencing instrument. This instrument, Alpha-1, will be placed at VisiGen and accelerate technology development. This instrument will be used for beta testing by researchers at the Baylor College of Medicine, Human Genome Sequencing Center (Houston, TX) and SeqWright, Inc. (Houston, TX). Feedback from personnel involved in these beta tests will be incorporated into the next phase of technology development.